CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit

A CYP2C19, 1.CYP2C19 technology, applied in the field of clinical molecular detection, can solve the problems of large template demand, high detection cost, and high demand for template quantity, and achieve the effect of high detection resolution and high detection sensitivity

Inactive Publication Date: 2016-08-17
BEIJING MICROREAD GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages of this method are: (1) Before the PCR reaction, DNA needs to be extracted from the sample to detect the quality and concentration, the operation is complicated, and there is a risk of confusing the sample
(2) When detecting multiple sites, one sample needs to perform multiple reactions, the detection cost is high, the demand for the amount of template is high, the operation intensity is high, and it is easy to cause operation errors and pollution
Generally speaking, the steps are cumbersome, the test period is long, and the cost is high, which is not conducive to large-scale clinical promotion
2. RFLP analysis method: Based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of

Method used

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  • CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit
  • CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit
  • CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: A kit for detecting CYP2C19, CYP2C9 and VKORC1 genotyping

[0099] This kit consists of PCR Master Mix, positive control DNA (normal person), negative control (sterilized water), and internal standard ROX500. The main components of PCR Master Mix include hot start DNA Taq enzyme (5U / μL, KAPA), UDG enzyme (5U / μL, NEB), 1.25×Buffer and primers for each site, etc. The dosage of each component is: when the reaction system is 20 μL, 1.25×Buffer is 16 μL, hot start DNA Taq enzyme is 0.4 μL, UDG enzyme is 0.04 μL, primer Mix is ​​0.58 μL (the final concentration of each primer

[0100] 100-200nM), template DNA or blood is 1-2μL, and sterilized water is added to 20μL.

[0101] The original primer sequences of the six SNP sites are as follows:

[0102] The base sequence of the primers at the 681G>A site of the CYP2C19*2 gene is as follows:

[0103] Forward wild-type primer: 5'-TTTCCCACTATCATTGATTATTTCCCG-3'

[0104] Forward mutant primer: 5'-TTTCCCACTATCATTGATTATT...

Embodiment 2

[0156] Example 2: Amplification method used in a kit for detecting CYP2C19, CYP2C9 and VKORC1 genotyping

[0157] Step 1: Extraction of Genomic DNA from Whole Blood

[0158] Take 200 μL of EDTA anticoagulated blood (sample A), and extract blood genomic DNA according to the instructions of the whole blood genomic DNA extraction kit. Measure the concentration of DNA with a spectrophotometer and dilute to 5-10ng / μL for use.

[0159] Step 2: PCR amplification reaction

[0160] 1. Aliquot PCR premix solution (completed in the reagent preparation area)

[0161] Shake and mix the PCR master mix solution (PCR Master Mix), and it is estimated that 4 detections will be performed, and each PCR reaction tube will be filled with 19 μL.

[0162] 2. Add template (completed in the specimen preparation area)

[0163] The detection templates are the blood and DNA of the above-mentioned patient A, and the templates, 1 μL of blood sample, 1 μL of DNA sample, 1 μL of positive control and 1 μL ...

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Abstract

The invention discloses a CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit. The amplification system comprises two forward primers and a reverse fluorescent primer of six SNPs (single nucleotide polymorphisms) respectively and can simultaneously amplify the six SNPs. The system is characterized by achieving one-tube amplification of three SNPs on the gene CYP2C19, two SNPs on the gene CYP2C9 and one SNP on the gene VKORC1 through multiple PCR (polymerase chain reaction). In particular, the amplification system can achieve direct amplification of blood and blood spot samples and dispenses with the step of extracting DNA (deoxyribonucleic acid). The amplification system can integrate the UDG-dUTP antipollution measure and can effectively prevent product pollution. The detection system is comprehensive in site detection, is simple and convenient to operate, has high specificity, high sensitivity and strong reliability, is low in cost and has the capacity of mass detection.

Description

technical field [0001] The invention relates to a simultaneous detection of CYP2C19, CYP2C9 and VKORC1 gene polymorphisms related to the drug metabolism rate of clopidogrel and warfarin based on multiple fluorescent PCR technology and capillary electrophoresis technology, which is an individualized method for clinical treatment of cardiovascular and other diseases It provides reference in medication and belongs to clinical molecular detection technology in the field of biomedicine. Background technique [0002] Cytochrome P450 superfamily (Cytochrome P450proteins, CYP) is a group of isoenzymes encoded by superfamily genes related to structure and function, which mainly exist in monooxygenases in liver and intestine. Free in the cytoplasm in prokaryotes, it is a soluble protein; in eukaryotes, as a membrane-bound protein, it is mainly distributed on the inner membrane of the endoplasmic reticulum, microsomes and mitochondria, and its main function is in the drug ( Such as cl...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16C12Q2537/143C12Q2565/125C12Q2545/113C12Q2521/531C12Q2563/107
Inventor 陈初光张晔张明珠王鑫
Owner BEIJING MICROREAD GENE TECH
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